雷帕霉素对缺氧损伤视网膜神经节细胞的保护作用及其机制 |
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引用本文: | 任静,秦波,邹畅,何靖,刘身文. 雷帕霉素对缺氧损伤视网膜神经节细胞的保护作用及其机制[J]. 中华实验眼科杂志, 2017, 0(5): 404-409. DOI: 10.3760/cma.j.issn.2095-0160.2017.05.004 |
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作者姓名: | 任静 秦波 邹畅 何靖 刘身文 |
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作者单位: | 1. 暨南大学第一临床医学院眼科,广州,510632;2. 518040,深圳市眼科医院 暨南大学附属深圳眼科医院深 圳大学眼视光学院深圳眼科学重点实验室深圳眼外伤治疗与干细胞定向分化公共服务平台;3. 518000,深圳市人民医院临床研究中心 |
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基金项目: | the Basic Research Project of Knowledge Innovation Program in Shenzhen (JCYJ20150402152130696)深圳市知识创新计划基础研究项目(JCYJ20150402152130696) |
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摘 要: | 背景 研究表明雷帕霉素(Rapa)可延缓人大脑细胞的衰老,改善大鼠局灶性脑缺血时脑组织代谢活动,对中枢神经细胞具有保护作用.视神经和视网膜神经节细胞(RGCs)属于中枢神经组织,但Rapa是否可对外伤后RGCs具有保护作用尚不清楚. 目的 探讨Rapa对氯化钴(CoCl2)诱导的缺氧损伤大鼠RGCs的保护作用,并探讨其可能的作用机制,为外伤性视神经病变(TON)的治疗提供新的思路.方法 用含体积分数10%胎牛血清的DMEM培养基培养大鼠RGC-5细胞,倒置相差显微镜下观察培养细胞的形态学变化.将细胞分为正常对照组及50、100、200、400和600 μmol/L CoCl2组,于细胞培养后24 h和48 h采用细胞生长分析系统检测各组细胞存活率.采用200 μmol/L CoCl2处理细胞以诱导建立RGC-5细胞缺氧损伤模型,然后将培养的细胞分为正常对照组、模型对照组和不同浓度Rapa干预组,Rapa干预组在缺氧模型细胞培养液中添加Rapa使其终浓度分别为0.1、0.4、1.6和6.4μmol/L,处理细胞24 h.采用细胞生长分析系统检测各组细胞的存活率;采用AnnexinV-FITC/PI双染流式细胞术检测各组细胞凋亡率;采用JC-1染色技术检测各组细胞线粒体跨膜电位(△ψm)的变化;采用实时荧光定量PCR法检测各组细胞促凋亡基因bax mRNA的相对表达量. 结果 200 μmol/L CoC12作用RGC-5细胞后24 h细胞相对存活率为(70.51±5.00)%,与正常对照组的(100.00±3.29)%比较,差异有统计学意义(P<0.01),成功构建细胞缺氧损伤模型.正常对照组、模型对照组和0.1、0.4、1.6、6.4 μmol/L Rapa干预组细胞存活率的总体比较差异有统计学意义(F=167.904,P=0.000),其中0.1μmol/L Rapa干预组细胞存活率明显高于模型对照组,差异有统计学意义(P<0.05).正常对照组、模型对照组和0.1μmol/L Rapa干预组细胞凋亡率分别为25.4%、37.7%和25.3%,细胞线粒体膜电位分别下降了0.4%、6.3%和1.4%.正常对照组、模型对照组和0.1 μmol/L Rapa干预组细胞中baxmRNA相对表达量分别为1.01±0.21、3.52±0.30和1.66±0.20,总体比较差异有统计学意义(F=88.034,P=0.000),其中模型对照组细胞中bax mRNA相对表达量均明显高于正常对照组和0.1μmol/L Rapa干预组,差异均有统计学意义(均P<0.05). 结论 Rapa可对CoC12诱导的缺氧RGC-5细胞发挥保护作用,其主要作用机制是下调细胞中促凋亡分子bax的表达,并提高RGC-5细胞存活率.
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关 键 词: | 雷帕霉素 视网膜神经节细胞 哺乳动物雷帕霉素靶蛋白 细胞凋亡 |
Protective effect of rapamycin on hypoxia-injuried retinal ganglion cells and its mechanism |
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Abstract: | Background Studies showed that rapamycin (Rapa) plays a protective effect on central nervous system by improving the aging of human brain tissue and ameliorating rat cerebral metabolism after ischemia.Optical nerve and retinal ganglion cells (RGCs) are central nerve,however,whether Rapa can protect hypoxia-injuried RGCs is still unclear.Objective This study was to explore the protective role of Rapa on hypoxia RGC-5,a rat RGC line,and its underlying mechanism in order to provide a new strategy for the treatment of traumatic optic neuropathy.Methods Rat RGC-5 cells were cultured using DMEM with 10% fetal bovine serum,and the cells were observed under the inverted phase contrast microscope.The cells were treated by 50,100,200,400 and 600 μmol/L CoC12 for 24 hours and 48 hours,respectively,and Clone Select Imager was employed to assess the survival rate.The CoC12-induced hypoxia cell models were established by adding 200 μmol/L CoCl2 in the medium for 24 hours,and then the 0.1,0.4,1.6,6.4 μmol/L Rapa was used to treat the models for 24 hours in the Rapa intervention group,respectively.The cells cultured by DMEM with 10% fetal bovine serum served as the normal control group.The survival rate of the cells was evaluated by Clone Select Imager;the apoptotic rate of the cells was assayed by AnnexinV-FITC/PI double-staining flow cytometry;JC-1 probe was used to detect the mitochondrial trans-membrane potential,and the expression of bax mRNA in the cells was detected by real-time fluorescence quantitative PCR.Results The survival rate of the cells was (70.51 ±5.00) % in the 200 μmol/L CoC12-treated group,which was significantly lower than (100.00±3.29)% in the normal control group (P<0.01).The survival rate of the cells was significantly different among the normal control group and 0.1,0.4,1.6,6.4 μmol/L Rapa intervention groups (F=167.904,P =0.000),and the survival rate was evidently higher in the 0.1 μmol/L Rapa intervention group than that in the model control group (P<0.05).The apoptotic rate was 25.4%,37.7% and 25.3%,while mitochondrial transmembrane potential reduced by 0.4%,6.3% and 1.4% in the normal control group,model control group and 0.1 μmol/L Rapa intervention group,respectively.The relative expression of bax mRNA in the cells was 1.01±0.21,3.52-±0.30 and 1.66±0.20 in the normal control group,model control group and 0.1 μmol/L Rapa intervention group,showing a significant difference among the groups (F =88.034,P =0.000),and the relative expression of bax mRNA in the model control group was considerably elavated in comparison with the normal control group and 0.1 μmol/L Rapa intervention group (both at P< 0.05).Conclusions Rapa protects the RGC-5 cells against CoC12-induced hypoxic damage primarily by down-regulating the expression of bax in the cells and improving the survival rate of RGC-5 cells in vitro. |
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Keywords: | Rapamycin Retinal ganglion cells Mammalian target of rapamycin Apoptosis |
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